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ATM-mediated stabilization of ZEB1 promotes DNA damage response and radioresistance through CHK1.

Zhang P, Wei Y, Wang L, Debeb BG, Yuan Y, Zhang J, Yuan J, Wang M, Chen D, Sun Y, Woodward WA, Liu Y, Dean DC, Liang H, Hu Y, Ang KK, Hung MC, Chen J, Ma L - Nat. Cell Biol. (2014)

Bottom Line: However, it is unclear whether EMT itself or specific EMT regulators play causal roles in these properties.Here we identify an EMT-inducing transcription factor, zinc finger E-box binding homeobox 1 (ZEB1), as a regulator of radiosensitivity and DNA damage response.These findings identify ZEB1 as an ATM substrate linking ATM to CHK1 and the mechanism underlying the association between EMT and radioresistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA.

ABSTRACT
Epithelial-mesenchymal transition (EMT) is associated with characteristics of breast cancer stem cells, including chemoresistance and radioresistance. However, it is unclear whether EMT itself or specific EMT regulators play causal roles in these properties. Here we identify an EMT-inducing transcription factor, zinc finger E-box binding homeobox 1 (ZEB1), as a regulator of radiosensitivity and DNA damage response. Radioresistant subpopulations of breast cancer cells derived from ionizing radiation exhibit hyperactivation of the kinase ATM and upregulation of ZEB1, and the latter promotes tumour cell radioresistance in vitro and in vivo. Mechanistically, ATM phosphorylates and stabilizes ZEB1 in response to DNA damage, ZEB1 in turn directly interacts with USP7 and enhances its ability to deubiquitylate and stabilize CHK1, thereby promoting homologous recombination-dependent DNA repair and resistance to radiation. These findings identify ZEB1 as an ATM substrate linking ATM to CHK1 and the mechanism underlying the association between EMT and radioresistance.

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ATM phosphorylates and stabilizes ZEB1(a) 293T cells were transfected with SFB-ZEB1 and treated with IR,followed by pull-down with streptavidin-sepharose beads and immunoblotting withantibodies to ATM and FLAG.(b) SUM159-P2 cells were transduced with ATM shRNA and treated withIR. Lysates were immunoblotted with antibodies to p-ATM, ATM, ZEB1, CHK1 andGAPDH.(c) SUM159-P2 cells with or without Ku55933 pretreatment (10μM, 1 hr) were treated with IR (6 Gy) and CHX (50 μg/ml),harvested at different time points, immunoprecipitated with the ZEB1 antibodyand immunoblotted with antibodies to p-S/TQ and ZEB1.(d) 293T cells were transfected with SFB-ZEB1 and treated with IR,followed by pull-down with streptavidin-sepharose beads and immunoblotting withantibodies to p-S/TQ and ZEB1.(e) Endogenous ZEB1 was immunoprecipitated from SUM159-P0 andSUM159-P2 cells and immunoblotted with antibodies to p-S/TQ and ZEB1.(f) Consensus ATM phosphorylation site on human ZEB1 (S585) andalignment with the conserved site on mouse, rat and Xenopus Zeb1.(g) 293T cells were transfected with wild-type, the S585A or S585Dmutant of SFB-ZEB1 and treated with IR, followed by pull-down withstreptavidin-sepharose beads and immunoblotting with antibodies to p-S/TQ andZEB1.(h) Immunopurified wild-type ZEB1 or the S585A mutant was incubatedwith immunopurified wild-type ATM or the kinase-dead (KD) mutant in kinasebuffer containing 32P-ATP. After reaction, proteins were resolved bySDS-PAGE and subjected to autoradiography and immunoblotting with antibodies toZEB1 and p-ATM. Purified GST-p53 was used as a positive control for ATM kinaseactivity.(i) HeLa cells were co-transfected with SFB-GFP and wild-type, theS585A or S585D mutant of SFB-ZEB1, treated with CHX with or without IR,harvested at different time points and immunoblotted with antibodies to FLAG.SFB-GFP serves as the control for transfection.(j) Clonogenic survival assays of SUM159-P0 cells transfected withwild-type ZEB1 or the mutant. n = 3 wells pergroup.Data in j are the mean of biological replicates from arepresentative experiment, and error bars indicate s.e.m. Statisticalsignificance was determined by a two-tailed, unpaired Student’st-test. The experiments were repeated 3 times. The sourcedata can be found in Supplementary Table 3. Uncropped images of blots are shown in Supplementary Figure7.
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Figure 7: ATM phosphorylates and stabilizes ZEB1(a) 293T cells were transfected with SFB-ZEB1 and treated with IR,followed by pull-down with streptavidin-sepharose beads and immunoblotting withantibodies to ATM and FLAG.(b) SUM159-P2 cells were transduced with ATM shRNA and treated withIR. Lysates were immunoblotted with antibodies to p-ATM, ATM, ZEB1, CHK1 andGAPDH.(c) SUM159-P2 cells with or without Ku55933 pretreatment (10μM, 1 hr) were treated with IR (6 Gy) and CHX (50 μg/ml),harvested at different time points, immunoprecipitated with the ZEB1 antibodyand immunoblotted with antibodies to p-S/TQ and ZEB1.(d) 293T cells were transfected with SFB-ZEB1 and treated with IR,followed by pull-down with streptavidin-sepharose beads and immunoblotting withantibodies to p-S/TQ and ZEB1.(e) Endogenous ZEB1 was immunoprecipitated from SUM159-P0 andSUM159-P2 cells and immunoblotted with antibodies to p-S/TQ and ZEB1.(f) Consensus ATM phosphorylation site on human ZEB1 (S585) andalignment with the conserved site on mouse, rat and Xenopus Zeb1.(g) 293T cells were transfected with wild-type, the S585A or S585Dmutant of SFB-ZEB1 and treated with IR, followed by pull-down withstreptavidin-sepharose beads and immunoblotting with antibodies to p-S/TQ andZEB1.(h) Immunopurified wild-type ZEB1 or the S585A mutant was incubatedwith immunopurified wild-type ATM or the kinase-dead (KD) mutant in kinasebuffer containing 32P-ATP. After reaction, proteins were resolved bySDS-PAGE and subjected to autoradiography and immunoblotting with antibodies toZEB1 and p-ATM. Purified GST-p53 was used as a positive control for ATM kinaseactivity.(i) HeLa cells were co-transfected with SFB-GFP and wild-type, theS585A or S585D mutant of SFB-ZEB1, treated with CHX with or without IR,harvested at different time points and immunoblotted with antibodies to FLAG.SFB-GFP serves as the control for transfection.(j) Clonogenic survival assays of SUM159-P0 cells transfected withwild-type ZEB1 or the mutant. n = 3 wells pergroup.Data in j are the mean of biological replicates from arepresentative experiment, and error bars indicate s.e.m. Statisticalsignificance was determined by a two-tailed, unpaired Student’st-test. The experiments were repeated 3 times. The sourcedata can be found in Supplementary Table 3. Uncropped images of blots are shown in Supplementary Figure7.

Mentions: We asked whether ZEB1 is regulated by ATM. Co-immunoprecipitationrevealed physical interaction of ZEB1 with ATM (Fig. 7a), whereas ATR showed no association with ZEB1 (Supplementary Fig. 5a).Moreover, depletion of ATM in SUM159-P2 cells significantly downregulated ZEB1and CHK1 proteins (Fig. 7b); in contrast,neither knockdown of ATR or treatment with the ATR inhibitor ETP-4646450 affected ZEB1 protein levels(Supplementary Fig. 5b,c). ATM substrates have a common S/T-Q motif. Analysis of the ZEB1protein sequence revealed one evolutionarily conserved S/T-Q motif encompassingserine 585. An inhibitor of ATM kinase, Ku55933, significantly decreased thestability of ZEB1 and reduced S/T-Q phosphorylation of ZEB1, as gauged by aphospho-S/TQ (p-S/TQ) antibody (Fig. 7c).Moreover, this phospho-S/TQ antibody detected much higher signals in ZEB1purified from irradiated 293T cells than that purified from non-irradiated cells(Fig. 7d). Consistently, S/T-Qphosphorylation of endogenous ZEB1 was significantly upregulated in SUM159-P2cells which displayed much higher levels of ATM phosphorylation than SUM159-P0cells (Fig. 7e).


ATM-mediated stabilization of ZEB1 promotes DNA damage response and radioresistance through CHK1.

Zhang P, Wei Y, Wang L, Debeb BG, Yuan Y, Zhang J, Yuan J, Wang M, Chen D, Sun Y, Woodward WA, Liu Y, Dean DC, Liang H, Hu Y, Ang KK, Hung MC, Chen J, Ma L - Nat. Cell Biol. (2014)

ATM phosphorylates and stabilizes ZEB1(a) 293T cells were transfected with SFB-ZEB1 and treated with IR,followed by pull-down with streptavidin-sepharose beads and immunoblotting withantibodies to ATM and FLAG.(b) SUM159-P2 cells were transduced with ATM shRNA and treated withIR. Lysates were immunoblotted with antibodies to p-ATM, ATM, ZEB1, CHK1 andGAPDH.(c) SUM159-P2 cells with or without Ku55933 pretreatment (10μM, 1 hr) were treated with IR (6 Gy) and CHX (50 μg/ml),harvested at different time points, immunoprecipitated with the ZEB1 antibodyand immunoblotted with antibodies to p-S/TQ and ZEB1.(d) 293T cells were transfected with SFB-ZEB1 and treated with IR,followed by pull-down with streptavidin-sepharose beads and immunoblotting withantibodies to p-S/TQ and ZEB1.(e) Endogenous ZEB1 was immunoprecipitated from SUM159-P0 andSUM159-P2 cells and immunoblotted with antibodies to p-S/TQ and ZEB1.(f) Consensus ATM phosphorylation site on human ZEB1 (S585) andalignment with the conserved site on mouse, rat and Xenopus Zeb1.(g) 293T cells were transfected with wild-type, the S585A or S585Dmutant of SFB-ZEB1 and treated with IR, followed by pull-down withstreptavidin-sepharose beads and immunoblotting with antibodies to p-S/TQ andZEB1.(h) Immunopurified wild-type ZEB1 or the S585A mutant was incubatedwith immunopurified wild-type ATM or the kinase-dead (KD) mutant in kinasebuffer containing 32P-ATP. After reaction, proteins were resolved bySDS-PAGE and subjected to autoradiography and immunoblotting with antibodies toZEB1 and p-ATM. Purified GST-p53 was used as a positive control for ATM kinaseactivity.(i) HeLa cells were co-transfected with SFB-GFP and wild-type, theS585A or S585D mutant of SFB-ZEB1, treated with CHX with or without IR,harvested at different time points and immunoblotted with antibodies to FLAG.SFB-GFP serves as the control for transfection.(j) Clonogenic survival assays of SUM159-P0 cells transfected withwild-type ZEB1 or the mutant. n = 3 wells pergroup.Data in j are the mean of biological replicates from arepresentative experiment, and error bars indicate s.e.m. Statisticalsignificance was determined by a two-tailed, unpaired Student’st-test. The experiments were repeated 3 times. The sourcedata can be found in Supplementary Table 3. Uncropped images of blots are shown in Supplementary Figure7.
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Figure 7: ATM phosphorylates and stabilizes ZEB1(a) 293T cells were transfected with SFB-ZEB1 and treated with IR,followed by pull-down with streptavidin-sepharose beads and immunoblotting withantibodies to ATM and FLAG.(b) SUM159-P2 cells were transduced with ATM shRNA and treated withIR. Lysates were immunoblotted with antibodies to p-ATM, ATM, ZEB1, CHK1 andGAPDH.(c) SUM159-P2 cells with or without Ku55933 pretreatment (10μM, 1 hr) were treated with IR (6 Gy) and CHX (50 μg/ml),harvested at different time points, immunoprecipitated with the ZEB1 antibodyand immunoblotted with antibodies to p-S/TQ and ZEB1.(d) 293T cells were transfected with SFB-ZEB1 and treated with IR,followed by pull-down with streptavidin-sepharose beads and immunoblotting withantibodies to p-S/TQ and ZEB1.(e) Endogenous ZEB1 was immunoprecipitated from SUM159-P0 andSUM159-P2 cells and immunoblotted with antibodies to p-S/TQ and ZEB1.(f) Consensus ATM phosphorylation site on human ZEB1 (S585) andalignment with the conserved site on mouse, rat and Xenopus Zeb1.(g) 293T cells were transfected with wild-type, the S585A or S585Dmutant of SFB-ZEB1 and treated with IR, followed by pull-down withstreptavidin-sepharose beads and immunoblotting with antibodies to p-S/TQ andZEB1.(h) Immunopurified wild-type ZEB1 or the S585A mutant was incubatedwith immunopurified wild-type ATM or the kinase-dead (KD) mutant in kinasebuffer containing 32P-ATP. After reaction, proteins were resolved bySDS-PAGE and subjected to autoradiography and immunoblotting with antibodies toZEB1 and p-ATM. Purified GST-p53 was used as a positive control for ATM kinaseactivity.(i) HeLa cells were co-transfected with SFB-GFP and wild-type, theS585A or S585D mutant of SFB-ZEB1, treated with CHX with or without IR,harvested at different time points and immunoblotted with antibodies to FLAG.SFB-GFP serves as the control for transfection.(j) Clonogenic survival assays of SUM159-P0 cells transfected withwild-type ZEB1 or the mutant. n = 3 wells pergroup.Data in j are the mean of biological replicates from arepresentative experiment, and error bars indicate s.e.m. Statisticalsignificance was determined by a two-tailed, unpaired Student’st-test. The experiments were repeated 3 times. The sourcedata can be found in Supplementary Table 3. Uncropped images of blots are shown in Supplementary Figure7.
Mentions: We asked whether ZEB1 is regulated by ATM. Co-immunoprecipitationrevealed physical interaction of ZEB1 with ATM (Fig. 7a), whereas ATR showed no association with ZEB1 (Supplementary Fig. 5a).Moreover, depletion of ATM in SUM159-P2 cells significantly downregulated ZEB1and CHK1 proteins (Fig. 7b); in contrast,neither knockdown of ATR or treatment with the ATR inhibitor ETP-4646450 affected ZEB1 protein levels(Supplementary Fig. 5b,c). ATM substrates have a common S/T-Q motif. Analysis of the ZEB1protein sequence revealed one evolutionarily conserved S/T-Q motif encompassingserine 585. An inhibitor of ATM kinase, Ku55933, significantly decreased thestability of ZEB1 and reduced S/T-Q phosphorylation of ZEB1, as gauged by aphospho-S/TQ (p-S/TQ) antibody (Fig. 7c).Moreover, this phospho-S/TQ antibody detected much higher signals in ZEB1purified from irradiated 293T cells than that purified from non-irradiated cells(Fig. 7d). Consistently, S/T-Qphosphorylation of endogenous ZEB1 was significantly upregulated in SUM159-P2cells which displayed much higher levels of ATM phosphorylation than SUM159-P0cells (Fig. 7e).

Bottom Line: However, it is unclear whether EMT itself or specific EMT regulators play causal roles in these properties.Here we identify an EMT-inducing transcription factor, zinc finger E-box binding homeobox 1 (ZEB1), as a regulator of radiosensitivity and DNA damage response.These findings identify ZEB1 as an ATM substrate linking ATM to CHK1 and the mechanism underlying the association between EMT and radioresistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA.

ABSTRACT
Epithelial-mesenchymal transition (EMT) is associated with characteristics of breast cancer stem cells, including chemoresistance and radioresistance. However, it is unclear whether EMT itself or specific EMT regulators play causal roles in these properties. Here we identify an EMT-inducing transcription factor, zinc finger E-box binding homeobox 1 (ZEB1), as a regulator of radiosensitivity and DNA damage response. Radioresistant subpopulations of breast cancer cells derived from ionizing radiation exhibit hyperactivation of the kinase ATM and upregulation of ZEB1, and the latter promotes tumour cell radioresistance in vitro and in vivo. Mechanistically, ATM phosphorylates and stabilizes ZEB1 in response to DNA damage, ZEB1 in turn directly interacts with USP7 and enhances its ability to deubiquitylate and stabilize CHK1, thereby promoting homologous recombination-dependent DNA repair and resistance to radiation. These findings identify ZEB1 as an ATM substrate linking ATM to CHK1 and the mechanism underlying the association between EMT and radioresistance.

Show MeSH
Related in: MedlinePlus